The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially-valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA, and polyethylene glycol (lithium acetate treatment) generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homolog of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus...

Electroporation enables delivering bionanomolecules, such as DNAs, RNAs, siRNAs, and morpholinos, into chick embryos in a spatially and temporally restricted fashion. Recent advances in electroporation techniques allowed us to deliver transgenes into the restricted area of the limb bud and to analyze the function of the enhancers in the limb field. Here, we describe the introduction of transgenes by electroporation in the limb field and its application on enhancer analysis.

Chicken embryo electroporation is a powerful tool used to identify and analyze enhancers involved in developmental gene regulation. In this chapter, the basic procedures and underlying principles of enhancer analysis using chicken embryo electroporation are described in the following steps: (1) identification of enhancers in a wide genomic region, (2) determination of the full enhancer region, (3) definition of the core enhancer regions, and (4) analysis of a functional transcription factor binding sequences in the core region...

One of the advantages of the avian embryo as an experimental model is its in ovo development and hence accessibility for genetic manipulation. Electroporation has been used extensively in the past to study gene function in chicken and quail embryos . Readily accessible tissues such as the neural tube, somites, and limb bud, in particular, have been targeted. However, more inaccessible tissues, such as the embryonic urogenital system , have proven more challenging to study. Here, we describe the use of in ovo electroporation of TOL2 vectors or RCASBP avian viral vectors for the rapid functional analysis of genes involved in avian sex determination and urogenital development ...

To elucidate a gene function, in vivo analysis is indispensable. We can carry out gain and loss of function experiment of a gene of interest by electroporation in ovo and ex ovo culture system on early-stage and advanced-stage chick embryos, respectively. In this section, we introduce in/ex ovo electroporation methods for the development of the chick central nervous system and visual system investigation.

Real-time four-dimensional (4D, xyzt) imaging of cultured avian embryos is an ideal method for investigating the complex movements of cells and tissues during early morphogenesis. While methods that transiently label cells, such as electroporation, are highly useful for dynamic imaging, they can also be limiting due to the number and type of cells that can be effectively targeted. In contrast, the heritable, stable, and long-term expression of a fluorescent protein driven by the exogenous promoter of a transgene overcomes these challenges...

Genome editing is driving a revolution in the biomedical sciences that carries the promise for future treatments of genetic diseases. The CRISPR/Cas9 system of RNA-guided genome editing has been successfully applied to modify the genome of a wide spectrum of organisms. We recently showed that this technique can be combined with in vivo electroporation to inhibit the function of genes of interest in somatic cells of the developing chicken embryo. We present here a simplified version of the previously described technique that leads to effective gene loss-of-function...

In biotechnology, medicine, and food processing, simple and reliable methods for cell membrane permeabilization are required for drug/gene delivery into the cells or for the inactivation of undesired microorganisms. Pulsed electric field treatment is among the most promising methods enabling both aims. The drawback in current technology is controllable large volume operation. To address this challenge, we have developed an experimental setup for flow through electroporation with online regulation of the flow rate with feedback control...

Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively...

Lassa virus (LASV) causes a severe hemorrhagic fever endemic throughout western Africa. Because of the ability to cause lethal disease in humans, limited treatment options, and potential as a bioweapon, the need for vaccines to prevent LASV epidemic is urgent. However, LASV vaccine development has been hindered by the lack of appropriate small animal models for efficacy evaluation independent of biosafety level four (BSL-4) facilities. Here we generated an LASV-glycoprotein precursor (GPC)-pseudotyped Human immunodeficiency virus containing firefly luciferase (Fluc) reporter gene as surrogate to develop a bioluminescent-imaging-based BALB/c mouse model for one-round infection under non-BSL-4 conditions, in which the bioluminescent intensity of Fluc was utilized as endpoint when evaluating vaccine efficacy...

During cortical development, neurons undergo polarization, oriented migration and layer-type differentiation. The biological and biochemical mechanisms underlying these processes are not completely understood. In neurons in culture we showed that IGF-1 receptor activation is important for growth cone assembly and axonal formation. However, the possible roles of the insulin like growth factor-1 receptor (IGF-1R) on neuronal differentiation and polarization in vivo in mammals have not yet been studied. Using in utero electroporation, we show here that the IGF-1R is essential for neocortical development...

High frequency irreversible electroporation (H-FIRE) is an emerging cancer therapy which uses bursts of short duration, alternating polarity, high voltage electrical pulses to focally ablate tumors. Here, we present a preliminary investigation of the combinatorial effects of H-FIRE and ionizing radiation. In vitro cell cultures were exposed to bursts of 500 ns pulses and single radiation doses of 2 or 20 Gy then analyzed for 14 days. H-FIRE and RT appear to induce different delayed cell death mechanisms and in all treatment groups, combinatorial therapy resulted in lower overall viabilities...

BACKGROUND: Generation of different neuronal subtypes during neocortical development is the most important step in the establishment of cortical cytoarchitecture. The transcription factor Satb2 is expressed in neocortical projection neurons that send their axons intracortically as opposed to Satb2-negative neurons that preferentially project to subcortical targets. NEW METHOD: In this report, we present a novel method to carry out large scale screening for molecules that control cell fate in the developing neocortex...

The combination of Irreversible Electroporation and Electrochemotherapy (IRECT) was well tolerated, safe, and had antitumor activity in this case study of a patient with lymph node metastases from gastric cancer. We therefore recommend the consideration of further clinical studies to investigate the treatment of cancerous tissue with IRECT.

Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a pre-spike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture...

In this study, a xenogeneic DNA vaccine encoding for human vascular endothelial growth factor receptor-2 (hVEGFR-2) was evaluated in two murine tumor models, the B16-F10 melanoma and the EO771 breast carcinoma model. The vaccine was administered by intradermal injection followed by electroporation. The immunogenicity and the biological efficacy of the vaccine were tested in (1) a prophylactic setting, (2) a therapeutic setting, and (3) a therapeutic setting combined with surgical removal of the primary tumor...